The goal of this investigation is to gain an understanding of the enzymatic mechanisms involved in mutagenesis during the replication of damaged DNA. Since the mutagenic effect of agents causing DNA damage may involve error-prone DNA replication and repair processes, the proposed studies will investigate the response of the DNA replication apparatus to specific lesions in template DNA and the induction of mutations that may be a consequence of repairing or bypassing DNA damage during replication. In order to study the mechanisms of mutagenesis during DNA replication at an enzymatic and molecular level, an in vitro system for the replication of ultraviolet-irradiated bacteriophage of DNA will be developed. Replication reactions will be carried out in extracts of gently-lysed Escherichia coli cells or in a reconstituted system of purified E. coli proteins, including RNA polymerase, DNA polymerase III, DNA binding protein, elongation factors I and III, and dnaZ protein. The system will be used to study the specific effects of proteins implicated in mutagenic processes, such as the E. coli rec A protein. The system will also provide a means to detect and purify previously unidentified proteins that are involved in the error-prone replication of lesion-containing DNA. Assays will be developed to assess mutation induction during the in vitro replication of lesion-containing DNA template.